Chick chorioallantoic membrane model to investigate role of migrasome in angiogenensis

The development of the vascular system is essential for embryonic development, including processes such as angiogenesis. Angiogenesis plays a critical role in many normal physiological and pathological processes. It is driven by a set of angiogenic proteins, including angiogenic growth factors, chemokines, and extracellular matrix proteins. Among various animal model systems, the chorioallantoic membrane (CAM), a specialized and highly vascularized tissue of the avian embryo, has proven to be a valuable tool for analyzing the angiogenic potential of candidate cells or factors. In this protocol, we provide detailed procedures for establishing the CAM model to evaluate the function and mechanism of migrasomes in embryonic angiogenesis. This includes the CAM nylon mesh assay and CAM ex vivo sprouting assay to assess CAM angiogenesis, as well as the observation, purification, and delivery of migrasomes. Additionally, we describe the generation of T4-KO-mCherry-KI embryos using the CRISPR system within the CAM tissue to investigate the role of migrasomes in angiogenesis.


INTRODUCTION
Angiogenesis is a progressive process in which new blood vessels are generated from pre-existing vasculature.To analyze the mechanisms underlying physiological and pathological angiogenesis, numerous in vivo angiogenic assays have been established using different species of laboratory animals, including mammals, birds, and zebrafish.In this manuscript, we will focus on major models of angiogenesis in the chick embryo.The use of chick embryo models for angiogenic studies is facilitated by a specialized respiratory tissue known as the chorioallantoic membrane (CAM).This structure rapidly expands, generating a rich vascular network that provides an interface for gas and waste exchange (Deryugina and Quigley 2008).The CAM is a relatively simple, quick, and low-cost model for studying tissue grafts, tumor growth and metastasis, drug delivery, toxicological analysis, and angiogenic and anti-angiogenic molecules.Moreover, because the chick embryo is naturally immunodeficient, it can receive transplantations from different tissues and species without eliciting immune responses (Ribatti 2017).
During chick embryo development, the allantois appears at approximately embryonic day 3.5 (E3.5d).The allantoic vesicle develops rapidly starting from embryonic day 4 (E4d) and reaches its peak at embryonic day 10 (E10d).This allantois fuses with the adjacent mesodermal layer of the chorion to form the CAM.The CAM vascularization undergoes three phases of development: the first phase involves sprouting and fusion to form the primary capillary plexus; the second phase, instead of sprouting, is characterized by predominantly intussusceptive angiogenesis, promoting capillary growth; and in the last phase, the growing pillars increase to form capillary meshes.Because immature blood vessels scattered in the mesoderm grow very rapidly until day 8-10 and give rise to a capillary plexus, we selected CAM samples at embryonic day 8-10 (Chen et al. 2021;Deryugina and Quigley 2008;Ribatti 2017).
Migrasomes are newly discovered organelles that form at the tips or intersections of retraction fibers (RFs).When a cell migrates, numerous retraction fibers are pulled out from the trailing edge of the cell, and large vesicles named migrasomes grow on these retraction fibers.These structures are released into the extracellular environment or taken up by surrounding cells, mediating the release of cytoplasmic contents and facilitating intercellular communication (Ma et al. 2015).
During zebrafish embryonic development, migrasomes are enriched with the chemokine CXCL12, which acts as a chemoattractant to guide the migration of dorsal forerunner cells (Jiang et al. 2019).Furthermore, migrasomes have been shown to mediate the lateral transfer of mRNA among cells (Zhu et al. 2021).Therefore, migrasomes have been proposed as organelles for cell-cell communication.However, research on the physiological functions of migrasomes is still in its early stages.
Based on recent advances in migrasome research and animal model studies of angiogenesis, we describe a series of assays using the CAM model to investigate the physiological functions of migrasomes in embryo development (Zhang et al. 2022).This may help expand the application of the chick CAM model in the study of angiogenesis.

Overview of the protocol
Given the nascent stage of exploration into the physiological functions of migrasomes, it remains uncertain whether migrasomes play a role in angiogenesis during embryonic development (Tan et al. 2023;Zhang et al. 2022).The CAM, serving as an animal model for the study of blood vessel formation, offers distinct advantages which we describe below.However, it is noteworthy that chicken embryos, belonging to the avian species and evolutionarily distant from mammals, render the chicken embryo animal model relatively specialized and niche in nature (Fishman et al. 2019).Consequently, the repertoire of established experimental systems related to chicken embryos remains rather limited.Therefore, it is imperative to establish and optimize numerous new methodologies utilizing the CAM model.In this paper, we present protocols primarily tailored to the research requirements of migrasomes, comprising five integral components: gene editing in chick embryos, observational systems in the CAM, migrasome purification and delivery in the CAM, and a CAM angiogenesis assessment system.

Advantages of the CAM model
The CAM model is a widely used experimental platform in diverse biomedical research disciplines, offering numerous advantages (Nowak-Sliwinska et al. 2014).It provides easy access to the availability of chick embryos and the straightforward accessibility of the CAM for manipulation and observation.Its high vascularity ensures a robust blood supply, making it ideal for studying angiogenesis, tumor growth, and drug delivery.Additionally, its transparency allows direct real-time monitoring, enabling observations of processes such as blood vessel formation, graft integration, and drug diffusion.
Furthermore, the CAM model offers an environment that closely resembles either in vivo or ex vivo conditions, facilitating the investigation of physiological processes and interactions with various substances or materials.Its cost-effectiveness makes it a financially sound choice for large-scale research projects compared to alternative animal models.Lastly, the ethical consideration of using chick embryos aligns with regulations on animal welfare, reducing the necessity for other vertebrate animals in experiments

Limitations of the CAM model
The CAM model is a widely used experimental model in angiogenesis research due to its accessibility, low cost, and ease of use.However, it does have some disadvantages that should be considered.First, species specificity: the CAM model is specific to avian species.This restricts its application when studying mammalspecific angiogenesis mechanisms.Second, limited availability of reagents, such as antibodies.Third, it is relatively difficult to genetically modify the CAM.Researchers should carefully weigh its advantages and disadvantages and complement their findings with other models.

SUMMARIZED PROCEDURE
All institutional and national guidelines for the care and use of laboratory animals were followed.All animal studies were conducted according to the guidelines from the Animal Care and Use Committee of Tsinghua University and Xiamen University.

Observation of migrasome formation in CAM in vivo
1 A square hole was cut in the eggshell of embryonic day 9 (CAM9d). 2 CAM was stained by wheat germ agglutinin (WGA).3 Visualize CAM in a glass holder under a confocal microscope.

Migrasome purification in CAM9d
1 Migrasome purification was performed by iodixanol sucrose density-gradient centrifugation using an Optiprep kit (LYSISO1, Sigma-Aldrich).CAMs were isolated from eight embryonic 9-day old chicken embryos (CAM9d), and then subjected to mechanical mincing. 2 The chopped-up CAMs were shakingly digested with collagenase II and trypsin.3 The samples were centrifuged to remove the cell bodies and cell fragments, and finally centrifuged to collect crude migrasome sediment.4 The crude migrasome sediment was fractionated at 150,000 g in a multistep Optiprep dilution gradient to get the purified migrasome fraction.

Migrasome delivery and CAM angiogenesis assay
1 Open the window of the eggshell of embryonic day 9 (CAM9d) so that the CAM can be observed.2 Preparation of Matrigel-or agarose-embedded migrasomes.3 The congealed mixture embedded in migrasomes was placed on the designated side of the CAM in vivo.4 Examine and visualize the newly formed vessels at the position of migrasome delivery.

The CAM nylon mesh assay
1 Prepare the nylon meshes (cut into 4.41 mm 2 per piece).2 Migrasome embedded by a Matrigel-collagen I mixture was placed onto the nylon mesh for polymerization.3 Polymerized meshes were placed onto the outer region of CAM8d and incubated.4 The newly formed vessels were examined and visualized with a photo microscope.
The CAM ex vivo sprouting assay 1 Preparations of Matrigel-Collagen I matrix mixture on 35 mm confocal dishes.2 Dissected CAMs (about 1 mm 2 ) from E8d were harvested and embedded in the matrix.3 After 24 h, drops of soft agarose with or without migrasomes were added on opposite sides of the CAM. 4 Visualize and analyze ECs sprouting from CAMs.

Generation of T4-KO-mCherry-KI embryos by the CRISPR system
1 TSPAN4-Gallus sgRNA was designed and inserted into the sgRNA expression cassettes of the pUC57 vector.2 Evaluation of the TSPAN4 knockout efficiency by mCherry knock-in system.3 Generation of the TSPAN4-KO chick embryos by microinjection of CRISPR system mRNA mixture (TSPAN4-sgRNAs, Cas9 and mCherry-KI) into chick embryos and then electroporation.4 Detection of TSPAN4 Knockout efficiency by FACS and IF.

PROCEDURE Preparations of fertilized chick eggs [TIMING 18 h-15 d]
1 Fertilized SPF eggs (Variety: White Leghorn; Cleanliness: SPF, cleaned and disinfected with ultraviolet light; Storage: stored in a constant temperature chamber at 14 ± 2 °C, no more than ten days) were bought from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd. 2 The eggs were incubated in a hatching incubator at 37.5 °C with 60%-70% humidity.Eggs were turned every five minutes.3 All SPF chick embryos used in the study were incubated from 18 hours to 15 days (E15d or CAM15d).

Observation of migrasome formation in CAM in vivo [TIMING 5 h]
Because opening the window will disturb the temperature, humidity, and atmospheric pressure of the chicken embryo in the egg, it should be ensured that the chicken embryo can still survive when we observe The CAM model in investigating migrasome in angiogenensis

PROTOCOL
the CAM.After numerous assay optimizations, we have successfully resolved the issue and established the assay as follows.
1 A square hole (about 1 × 1 cm 2 ) was cut in the eggshell of embryonic day 9 (CAM9d). 2 WGA was diluted in 1× PBS (1:500, 200 μL 1× PBS) and then this mixture was added to the top of the CAM. 3 Staining for 20 min at 37.5 °C in a humidified incubator with 70% humidity.4 The egg was placed on a holder with the hole in direct contact with a cover glass, so that the weight of the egg held the CAM in tight contact with the cover glass.5 Last, the CAM was visualized under a confocal microscope for about 4 h.

Migrasome purification [TIMING 6 h]
Migrasome purification was performed by iodixanol sucrose density-gradient centrifugation using an Optiprep kit (LYSISO1, Sigma-Aldrich), the detailed procedure is shown below. 1 CAMs were isolated from eight E9d chicken embryos, and then subjected to mechanical mincing. 2 The chopped-up CAMs were shakingly digested with collagenase II for 30 min at 37 °C.3 Centrifugation, 1000 g, 5 min, got the sediment.4 The above sediment was treated with trypsin for another 20 min at 37 °C.5 The samples were centrifuged at 1,000 g for 5 min at 4 °C to remove the cell bodies and get supernatant.6 The above supernatant was followed by 4,000 g for 20 min at 4 °C to remove the cell fragments and get the supernatant.7 The above supernatant was finally centrifuged at 20,000 g for 20 min at 4 °C to get the sediment.8 The pellet containing the crude migrasome fraction was resuspended and lysed in extraction buffer (Sigma-Aldrich) and then fractionated at 150,000 g for 4 h at 4 °C in a multistep Optiprep dilution gradient.The detail gradient was: 3%, 5%, 8%, 12%, 16%, 19% (sample), 22.5% and 27%.9 After centrifugation, the fraction formed, and the migrasome was enriched at fractions 3-5. 10 Fractions were collected and added to 500 μL PBS.
Centrifugation was then performed at 20,000 g for 30 min at 4 °C to collect migrasome.11 The pellet was washed once with PBS and centrifuged at 4 °C, 2,000 g for 10 min.12 The supernatant was collected and centrifuged at 4 °C, 20,000 g for 30 min to obtain migrasomes for microscope observation or delivery.

Migrasome delivery onto CAM [TIMING 3 d]
1 Fertilized chicken eggs were incubated at 37.5 °C for 9 days and then opened so that the CAM could be observed.2 Preparation of migrasome embedded matrix.Firstly, 30 μg of purified migrasomes (from CAM of chicken embryos at E9d) were resuspended in 3 μL 1× PBS. 3 Resuspended migrasomes were mixed with 5 μL 2% low-melting-point agarose.As the control, 30 μg cell body isolated from CAM was mixed with 5 μL 2% low-melting-point agarose.4 The congealed mixture was placed on the designated side of the CAM in vivo for about three days.5 Three days later, numerous allantoic vessels developed in a "spoked-wheel" pattern.Then images were immediately captured by a stereoscope or confocal microscope.The density or number of newly formed capillaries in the CAM was quantified with Image J v1.8.0.112 or Image-Pro Plus software (Media Cybernetics).

The CAM nylon mesh assay [TIMING 5 d]
Since directly observing capillary growth towards migrasomes may be prone to artifacts, we carried out the Matrigel invasion assay, in which a nylon mesh coated by a collagen I-Matrigel matrix with or without migrasomes is placed on top of the CAMs, and neovascularization occurs upward and against gravity to invade the matrix.New blood vessels are induced to grow upright into the matrix from the underlying CAM, and therefore can be clearly discriminated from the background vascular network, providing a clear advantage for angiogenesis scoring.This represents a modification of the CAM angiogenesis method to offer a straightforward approach to quantifying angiogenesis.1 Preparation of nylon meshes.The nylon meshes (TS207-050, SK, pore size 300 μm, China) were cut into 4.41 mm 2 pieces, then autoclaved for 10 min and vacuum-dried.2 Open the window of the eggshell on chick embryonic 8d to expose the CAM. 3 Prepare the nylon mesh-matrix-migrasome polymerization mixture.10 μL Matrigel and 5 μL collagen I mixed either with 5 uL cell body from CAM (as control) or with 5 μL migrasomes (10 μg/μL) were placed onto the nylon mesh and incubated at 37 °C for 30 min and at 4 °C for 1 h for polymerization.4 The polymerized mixture was placed onto the outer region of CAM8d and incubated for five days.
5 The newly formed vessels were examined and visualized with a photo microscope (Leica EZ4W) or by Nikon Ti2-E.6 The density or number of newly formed capillaries in the CAM was quantified with Image J v1.8.0.112 or Image-Pro Plus software (Media Cybernetics).

CAM ex vivo sprouting assay [TIMING 3-6 d]
1 Depolymerization of matrigel in 4 °C 2 Matrix containing 150 μL Matrigel and 30 μL collagen I was mixed on ice and then added into 3.5 mm confocal dishes and incubated at 37 °C for 1 h for polymerization.3 Dissected CAMs (about 1 mm 2 ) from CAM9d were harvested and embedded in the matrix for 24 h without any medium in it.4 24 h later, add about 50 μL complete ECM to keep the humidity of CAM in the dish.
[CAUTION!] Matrigel-Collagen I Matrix preparation must be on the ice to keep the matrigel liquid state.Before dissected CAMs embedded in the matrix for 24 h, we should not add any buffer or medium in the culture dish to guarantee CAM adherence to the matrix.5 Drops of low melt agarose with or without migrasomes were added on opposite sides of the CAM for another 2-5 days.6 After 2-5 days of culture, ECs sprouting from CAMs were visualized by Nikon Ti2-E and analyzed by Nikon NIS-elements.7 Post-treated CAMs were stained by ASM1 and Actin, then the samples were visualized by Nikon A1 confocal microscopy to assess the role of migrasomes in chemo-attraction of ECs and tip-cell filopodia formation.

Generation of T4-KO-mCherry-KI embryos by the CRISPR system [TIMING 8 d]
Cas9 nickase and paired sgRNAs can be used to efficiently modify the genome of embryos with minimal off-target damage.This protocol describes the design and construction of paired sgRNAs plasmids, and the preparation of sgRNAs and Cas9 mRNAs for the injection of gastrulating chick embryos (E18h).

Part 1: Construction of pUC57-TSPAN4-sgRNA plasmids by the CRISPR system
Cas9 nickase and paired sgRNAs can be used to efficiently modify the genome of embryos with minimal off-target damage.This protocol describes the design and construction of pUC57-and TSPAN4-sgRNA plasmids.
1 Design of paired sgRNA oligos as manual.Order oligos as below.
[CAUTION!]For each sgRNA, the 5'-GGN (19) GG motif is preferred, and 5'-GN (20) GG or 5'-N (21) GG are also satisfactory.BLAT or BLAST, the sgRNA target sites in UCSC or ENSEMBL genome browsers to find those with few or no highly related sites in the genome.

Part 4: Generation of T4-KO-mCherry-KI plasmid and mRNA
Since there are no commercially available antibodies to detect TSPAN4, to evaluate the TSPAN4 knockout efficiency, mCherry was inserted into TSPAN4 at the position targeted by the sgRNA by mCherry-KI plasmid: T4-KO-mcherry-KI plasmid (Zhang et al. 2022).
1 Plasmid extraction and purification.
Part 5: Microinjection of the mRNA mixture into chick embryonic 18 h 1 Cut a square hole (0.6 × 0.6 cm 2 ) in the eggshell of gastrulating chick embryos (E18h). 2 Thaw aliquots of the Cas9 mRNA, TSPAN4-KO-sgRNAs, and T4-KO-mcherry-KI mRNA on ice.Dilute the above mRNA mixture with EmbryoMax® Injection Buffer to a concentration of 30 ng/μL Cas9 mRNA, 10 ng/μL TSPAN4-KO-sgRNAs mRNA, 10 ng/μL T4-KO-mcherry-KI mRNA, the total volume is 10 μL.Pipette the mixture up and down several times.3 Centrifuge at 4 °C for 1 min at top speed, and carefully transfer the above 10 μL supernatant into chick embryos at Hamburger Hamilton stage 4 (HH4, embryonic 18 h) using a glass capillary with a tip diameter of 0.1 mm. 4 Chick embryos were then electroporated using previously described techniques (Sauka-Spengler and Barembaum 2008).5 The hole was covered by Parafilm and the chick embryos were incubated at 37.5 °C in the air under 70% humidity.6 Knockout effciency were confirmed by direct FACS for mCherry-positive signal sorting and by in vivo imaging for mCherry-positive signal detection.

ANTICIPATED RESULTS
1 Following the protocol, we first designed an imaging protocol.Figure 1A shows the detailed operating process.The CAM stained by WGA was imaged and the migrasome formation by WGA high has been shown in Fig. 1B.
2 Since we have found the migrasome in CAM tissue, to detect the function of migrasome, we established the protocol for migrasome isolation and purification from CAM tissue.Figure 2A shows the detailed operating process.The purity of the isolated migrasomes in Fig. 2B was analyzed by IF and by western blot for various migrasome markers The CAM model in investigating migrasome in angiogenensis

PROTOCOL
we reported previously (Zhang et al. 2022).We found that the isolated migrasomes have the characteristic morphological features of migrasomes, and migrasome markers are highly enriched in the preparation.3 After we got the high-purity migrasome from CAM tissue, we delivered the migrasome to CAM to detect its function in angiogenesis.The detailed operating process is shown in Fig. 3A.The migrasome embedded into low melting agarose was added into CAM9d， three days later, We found that migrasomes significantly promoted capillary formation in this assay in contrast to the control (Figs.3B and 3C).
4 Since the migrasome embedded by agarose brings more or less nearby vessels hypoxia and gives the impression of a higher capillary density.We established an invasion assay named a nylon mesh assay where the growth of new vessels occurs upward and against gravity by invading a new matrix and independent of the plane of the CAM, this protocol may be far more desirable and less prone to artifacts.Figure 4A shows the diagram of how to operate the process.Figures 4B and 4C show the results when nylon mesh coated by a collagen I-Matrigel matrix with migrasomes was placed on top of the CAMs, the neovascularization upward and against gravity significantly promoted capillary The CAM model in investigating migrasome in angiogenensis PROTOCOL formation contrast to the matrix without the migrasome.5 Another assay to estimate the pro-or antiangiogenic ability of the migrasome is the CAM ex vivo sprouting assay.Figure 5A shows the detailed operating process.We found that five days after adding the soft agarose, there were many cells on the side of the CAM close to the migrasomecontaining agarose, but not on the side close to the control agarose.Cells on the migrasome side migrated much further from the edge of the CAM compared with cells on the control side.Moreover, there were significantly more EC sprouting events and elongated filopodia on the tip cell marked by ASM1 and actin on the migrasome side than on the control side (Figs.5B-5E).6 TSPAN4 is essential for the migrasome formation.
To detect whether the migrasome elimination The CAM model in investigating migrasome in angiogenensis

PROTOCOL
where TSPAN4 has been successfully knocked out.
Figure 6B shows that TSPAN4 knockout efficiency is more than 70%.groups for their helpful discussions and suggestions.We are grateful to the members of the Zhihua Liu groups for their helpful discussions and suggestions about the CRISPR-Cas9 system.This research was supported by the Ministry of Science and Technology of the People's Republic of China (grant no.2017YFA0503404), the National Natural Science Foundation of China (31790401, 92054301 and 32030023), and the Beijing Municipal Science & Technology Commission (Z201100005320019 and Z211100003321002) and internal grant from Tsinghua university (2030 plan).We thank the State Key Laboratory of Membrane Biology for facility support and assistance with spinning disk microscopy.We would like to acknowledge the assistance of SLSTU-Nikon Biological Imaging Center for assistance with using the NIKON A1RSiHD25 laser scanning confocal microscope and data analysis by NLS element software.

Compliance with Ethical Standards
Conflict of interest Li Yu is the scientific founder of Migrasome therapeutics Ltd, and Cuifang Zhang, Helen He, Shuyao Yin and Mingyi Gao declare that they have no conflict of interest.
Human and animal rights and informed consent All institutional and national guidelines for the care and use of laboratory animals were followed.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The CAM model in investigating migrasome in angiogenensis PROTOCOL

Fig. 1
Fig. 1 Observation of migrasome formation in the CAM in vivo.A Diagram of migrasome observation procedure in CAM.B Images of migrasomes formation of WGA high cell in CAM9d in vivo level.Left: confocal image of CAM stained by WGA in vivo level; scale bar, 8 μm.Right: time-lapse of migrasomes formation in CAM9d in vivo level; scale bar, 5 μm

Fig. 2 Fig. 3
Fig. 2 Migrasome purification from CAM9d tissue.A Diagram of migrasome from the procedure from CAM9d tissue.B Migration purity identification.Left panel: images of migrasomes stained by WGA purified from CAM9d; scale bar, 5 μm.Right panel: Western blot analysis of isolated CAM9d migrasomes with the indicated antibodies

Fig. 4 Fig. 5
Fig.4The CAM nylon mesh assay.A Diagram of migrasome CAM nylon mesh assay.B Migrasomes were delivered to CAM9d by nylon mesh, and newly formed capillaries on nylon mesh were visualized when the migrasome was added.Scale bar, 500 μm.C Newly formed capillaries onto nylon mesh were quantified.Error bar, ±SEM; n = 15 fields from three independent experiments; P values, two-tailed, unpaired t-test, P < 0.0001